Scanning Electron Microscopy

Volume 1986 Number 4 Article 37

8-23-1986

The Biomineralization in Social (Vespinae): The Presence of Statoliths

J. S. Ishay Tel Aviv University

T. Shimony Tel Aviv University

L. Arcan Agriculture Research Organization

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Recommended Citation Ishay, J. S.; Shimony, T.; and Arcan, L. (1986) "The Biomineralization in Social Wasps (Vespinae): The Presence of Statoliths," Scanning Electron Microscopy: Vol. 1986 : No. 4 , Article 37. Available at: https://digitalcommons.usu.edu/electron/vol1986/iss4/37

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THE BI0MINERALIZATI0NIN SOCIALWASPS (VESPINAE): THE PRESENCEOF STATOLITHS J.S. Ishay(*)l, T. (Benshalom) Shimony1 and L. Arcan2 loepartment of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv 69978 2 and Volcani Center, Agriculture Research Organization, Beit Dagan 50250, Israel

(Received for publication March 03, 1986, and in revised form August 23, 1986)

Abstract Introduction In social wasps granular aggregates occur Only a few members of the Insecta have a mainly in the head region and the body integu­ developed statocyst: A cuticular nodule behind ment. In the former location they are present the eyes called Palmen's organ is found in the both on the outer and inner side of the frons. head of the adult and larva of Ephemeroptera; On the outer side of the frons, in a groove tra­ in Dorymyrmex() there is a stato­ versing its center, there is a deep pit sur­ cyst above the coxa on the metathorax which is rounded by a membrane which forms a cyst-like an invagination of the cuticle, lined with tac­ sac. In the pit of the Vespa orientalis tile hairs and possessing sand grains within (Linne, 1771), there are aggregates comprislng the cavity. These organs probably function as sllicon (Sl), potassium (K), calclum (Ca) and gravity receptors (Marcus, 1956; Chapman, 1969). iron (Fe), which are arranged in morula-llke In the order Hymenoptera (and others) some fashlon. A granular material is secreted at the species are colored; ln the social wasps base of sensory hairs (sensilla), distributed on (Vespinae), there is a predominantly yellow to both sides of the groove, as well as along their green color found in the cuticula spread over shafts. Aggregates of sillcon and calcium are various parts of the body (Kemper and Dohring, also present on the inner side of the frons. 1967; Guiglla, 1972; Spradbery, 1973; Edwards, The integumental aggregates are comprised 1980), which ls of taxonomlc lmportance: it is of yellow pigment granules. In the abdominal also belleved to serve as a warning signal region these granules, whlch are concentrated (Keeton, 1967). Such pigmented cuticular areas in yellow stripes, assume a quasi-cylindrical in Hymenoptera (as well as the colored wings in shape, measuring 0.6 - 0.8 µmin length and 0.3 Lepidoptera) are formed by an infolding of the -0.4 µmin diameter. Their structure is affect­ cuticula inside and the pigment aggregates are ed by caste, age, exposure to sunlight, and within granules composed of pteridines within drugs lntroduced through feeding. Feeding on the epidermal cells or above them, unlike the colchlcine induces degeneration of the granules, melanin which is embedded inside the cuticula whereas feedlng on xanthines renders them re­ (Becker, 1937;; Desclmon, 1965; Harmsen, 1966; fractory to the destructive action of colchl­ Ziegler and Harmsen, 1969). These pigmented cine. Thelr main metalllc element is K. areas in social wasps have photoconductive as We postulate that 1) the granules on both well as thermoconductive properties (Croitoru sldes of the frons are part of a tympanlc and et al., 1978; !shay and Croitoru, 1978; !shay static organ; 2) the yellow granules (YG) dis­ et al., 1982). Slmilar, although not identlcal, persed all over the external surface of the body pigmented granules have also been observed in possess properties of organic semiconductors; honeybees (J.S. !shay, unpublished observation). 3) land 2 are piezoelectric, and are involved Static sense organs composed of a hollow both in gravlty orlentation and transposition statocyst surrounded by sensory hairs and con­ from photo to geotaxis. tainlng statoliths have been discerned in the heads of various species of Vesplnae (!shay et KEYWORDS: Statocysts and Statoliths in ; al., 1983; !shay and Shlmony, 1986). The pre­ Yellow Pigment Granules; Photo- and Thermoelect­ sent paper provides data on the variety of ric Propertles; Biomineralization; Bioaggregates granules encountered In social wasps, and also containing Si, K, Ca, Fe; Gravity Orientation in speculates on their probable functions. Insects. Materials and Methods (*)Address for correspondence: Jacob S. !shay, Dept. of Physiology and The majority of the observations were made Pharmacology, Sackler Faculty of Medicine, Tel on the adult population (workers, queens Aviv University, Ramat Aviv, Tel Aviv 69978, Israel Phone No. (03) 420 863

1619 !shay, J.S., (Benshalom) Shimony, T., Arcan, L. and males) of the Vespinae species: on Vespa Thln sections were stained wlth l.5°/o orientalis, collected from natural nests uranyl acetate and 0.3°/o lead citrate. around the Tel Aviv area in Israel, and on L Micrographs were obtained by a JEOL 1008 crabro (Linne 175B), Paravespula germanica electron microscope. (Fabricius 1793), media (Retzius Changes in the size and shape of the yellow l7B3) and~ saxonica (Fabr. 1793) collected pigment granules were evaluated after one of in Germany, as well as many other species of the following treatments or conditions: Vespinae and (!shay and Shimony, a) exposure of hornet workers to continuous l9B6). The used for experiments were light for 14 days; b) the effect of age, i.e., maintained in artificial breeding boxes (ABBs) the difference between young hornets (0-24 h), (!shay and Sadeh, 1975) in groups of 5-20, and adults (several weeks old), and 'old' queens kept on a regular diet of 30°/o sucrose solu­ (one year of age); c) addition to the diet of tion and morsels of meat (for details, see J. caffeine, theophylline, allopurinol, or purine I shay, 1964). alone (in a dosage of about 5 µg/hornet/day) Hornets and wasps were killed by freezing or together with colchicine for two weeks; d) and stored until processing. Specimens were deviation from the normal pattern due to an first observed under light microscopy for pre­ aberration caused either by excessive yellow liminary location of specific areas. pigment, or by its absence. Scanning electron microscopy (SEM) and energy The photoelectric properties of the yellow dispersive x-ray analysis (EDX) abdominal stripes in situ (i.e., when connected Microstructural investigation as well as to the intact body), or of the separated yellow associated chemical information were carried pigment granules, were examined by observing out by JEOL-35 SEM, Cambridge Stereoscan 180S the change in resistance under illumination and Energy Dispersive X-ray Analysis System - and/or in the dark. This was achieved by PROXANIII equipped with a Si (Li) - Sl2.5-4 exposing the hornet after each measurement to high resolution detector. a low temperature (approximately 0°C), for For microstructural observation, the spec­ 5-15 min. The change in resistance upon imens were attached to aluminium or copper illumination was measured with an integral stubs with silver paint and sputter-coated by white light from incandescent bulbs of 100 W a 200-300 nm gold layer. and 220 Vat a distance of 20 cm from the Parts of the internal surfaces in front specimen. The change in resistance as a were investigated after fixation for a minimum function of illumination with white light was period of 4 h with 2.5°/o glutaraldehyde in measured in two ways: l) during 15-20 min of O.lM phosphate buffer pH 7.2, dehydrated through irradiation with light (until a saturation graded ethanols, then critical point dried (CPD) level was attained), and 2) by further exposure from liquid carbon dioxide and carbon and/or to a light/dark rhythm every 2 min (i.e., gold-sputtered. intermittent illumination). Control experiments Location of a number of elements presented were made to ascertain that the electrical in the frons area was investigated by x-ray connection, i.e. the silver paint, tungsten microanalysis (EDX) on unfixed as well as on wires or the glass were not in any way photo­ fixed tissues. Semi-quantitative measurements conductive. (For further details, see !shay were performed on YGs using gelatin blocks as and Croitoru, 1978; I shay et al., 1980). an organic matrix. Methods for evaluating the thermoelectric Stereo pictures at low and high magnifi­ properties, thermoelectric (Seebeck) effect cation were performed in order to check the and electrical capacitance of the yellow versus proper geometry for x-ray analysis. Minute brown stripes of the hornet and cuticle parts of the above specimens (about 1-2 mm2) were also described earlier (Shimony and !shay, were tailored accordingly under a light micro­ l98la,b; !shay et al., 1982a; Shimony and scope and attached to a carbon stub by carbon !shay, 1984). In this study individual hornets paint. The EDXwas carried out at l5kV-20kV were attached with glue to a microscope slide accelerating voltage with a 50 and 100 sec between two silver paint electrodes. The (lifetime) counting time for each analysis. yellow stripes on the abdominal segments were Occasionally, a very thin layer of gold, connected to the electrodes with thin tungsten insufficient to obscure the EDXpeaks being wires. The ohmic properties of the contacts studied, was necessary in order to suppress and their stability with time were ascertained charging effects on some interesting areas. (!shay and Croitoru, 1978). The relative Transmission Electron Microscopy (TEM) humidity in the test room was maintained at For transmission electron microscopy, about 75°/o (± 5°/o). Measurements of the pieces from the head (frons area) were dis­ electrical resistance of the cuticle were taken sected out of fresh specimens and fixed in in total darkness in a thermostat in which the 5°/o glutaraldehyde buffered with 0.1 M temperature was gradually altered within a cacodylate buffer (pH 7.2). After fixation range as needed, and the electric resistivity for 24 hat 4°C, the material was washed in was gauged with an electrometer (Keithly Digi­ buffer and secondarily fixed for 2 h in 2°/o tal Electrometer, Model 616, which served as osmium tetroxide buffered with cacodylate. an ohmmeter) within resistance values of 0.3 Dehydration was accomplished with an ethanol x 107 Q to 7.8 x 107 Q. Earlier studies series followed by embedding in Epon Bl2. also described the electrical resistance of the hornet cuticle and changes in resistance

1620 Biomineralization in Social Wasps induced by xanthines ( I shay et al., 1982b; 10-20 µm. Similar groups of sensilla, although Rosenzweig et al., 1985). of different dimensions, were discerned on the heads of hornets and wasps of all the species Results examined. Elemental analysis (EDX) revealed that SEMAND EDX on the center side of the frons aggregates in the cyst-like sac region as well On the frons of V. orientalis' workers, a as those at the base of the sensilla are centrally-traversingcanal called the frontal composed of Si, K, Ca, Ti and Fe, as suture (or coronal suture) can be seen principal inorganic elements . The relative (Edwards, 1980), which is divided into three proportions of these elements have not yet been distinct structures: l) The upper section; 2) defined. We have focussed our efforts on the pit area ; 3) the lower portion. qualitative aspects. An evident fact: in The upper section, situated close to the younger hornets (workers) there was a higher median ocellus, is rather shallow and essen­ concentration in Ca versus Si (see J.S. !shay tially is comprised of a slightly recessed et al., 1983) (Fig. Sa, b, c). Fig. 6a band devoid of sensilla. A pit can be found presents a view of the front of the head of a at the middle part of this structure in the hornet displaying the frons region. Fig. 6b form of an elliptic well approximately 30 µm shows a cross-section in the tegumentary deep, 60-70 µm long (externally) and 30-40 µm layer adjacent to the ocelli. In the upper in maximal width (Fig. l). part a separate mass is observable - the On the underlying surface of the frons the layered cuticle - and underneath it a layer of margins of this well protrude, similar to a YGs are encased in delicate membranes. cone. As a continuation of the well, the Thickness of the yellow granule layer in this bottom of the canal elevates to a depth of only region (see arrow) is 15-20 µm and the metal- a few µm while its width increases. The upper lic element in these granules is K. Our EDS part of the canal is not discernible in L analyses were carried out at a qualitative crabro, D. media and P. germanica, while in D. level rather than quantitative. However, for ~caneither the upper nor the lower part the YGs semiquantitative data were obtained of the canal is evident (although the well is from gelatine blocks in which the yellow pig­ visible). In L orientali's there is a secre­ ments, separated from the tissue matrix as well tion from the upper rim and borders of the as standards, were embedded. Potassium (60°/•- well, which accumulates in the following forms: 700/0) and phosphorus (10°/•-30°/•) were the a) as a thin smooth and rather solid membrane principal elements detected in the x-ray spectra. composed of an amorphous material which lines A transmission electron micrograph showing the the inner surface of the well and also 'enve­ base of a sensillum and a layer of yellow lops' like a cyst wall sac, with a pore at its granules on the inner side of the frons is center (Fig. 1). In the well, alongside both presented (see Fig. 7) (J.S. I shay et al., in its margins, there are numerous spinules and preparation). Underneath this membrane is an crests which at times anastomose. This cyst­ additional, more internal membrane which is like sac is located mainly in the deepest part part of the layer lining the space and pro­ of the well; b) as a granular accretion shaped ducing the 'strings' (see below). Under- like a mushroom or berry within the cyst-like neath the frons and ocular sinus in all species sac (Fig. 2). of Vespinae examined there is a 'space' con­ At times there are several accretions which taining numerous strings (Fig. 6c). In L are of varying sizes and shapes fastened to orientalis the 'space' is harp-shaped and the borders of the well. There is an adhesive comprises an acoustic box (!shay and Shimony, secretion which holds the granules together 1986). Inside the acoustic box there are some within the sac as well as on and above the 120-150 strings stretched across its width margins of the well. from the frons to the lining above the Sensory hairs. There are three clear protocerebrum (Fig. 6d). The individual categories of sensory hairs (sensilla), which strings, arranged helically, are about 10-50 extend over the entire length of the canal µm apart with a length usually in inverse along the borders, and across the frons: l) relation to their thickness. The short strings The marginal sensilla of the canal, which have (see Fig. 6b) are 100-120 µm long and 8-15 µm accretions of Ca at the base, are 10-30 µm long in diameter, whereas the long strings (See Fig. and 1-3 µm thick at the base (Fig. 3). Occa­ 6c) attain 600-700 µmin length and 3-7 µmin sionally, the secretion accumulates not only diameter. When detached from their hold on at the base but also along the shaft of the the walls, they tend to coil and abbreviate sensillum, and comprises a single bead or a like a spring (Fig. 6e). In V. orientalis string of several beads of secretion over its the entire frons plate is studded (from the length (Fig. 4 for P. germanica). 2) The border inside) with the YGs. Each granule is about sensilla of the frons, which number 12-16, are 0.5 µm long and 0.4 µmin diameter (See Fig. filiform and straight, about 100 µm long and 6f). A cross-section of one of the strings 10 µm thick at the base, situated about 100 µm seen by TEMis presented in Figs. 8, 9). A apart. 3) The ankyroid sensilla arranged section of an axon bundle originating from the across the frons above the region of the canal protocerebrum and entering the acoustic box is (below the medium ocellus) whose length ranges shown ( Fig. 10). between 200-300 µm with a thickness between

7627 !shay, J.S., (Benshalom) Shimony, T., Arcan, L.

Flg. 3. Sensllla ln the frons of a V. orientalls worker. Prominently dlsplayed are some sensilla with accretion 'beads' close to thelr base. The accretlons contaln mainly Ca. l.l.ll..:.._J_. Enlarged region of a L orientalis Bar= 10 µm. worker in area of the well. The membrane covering the well appears 1n the center of the picture. Above the membrane, there are accre­ tions of a granular material accumulated at the well's margins (see area marked by arrow no. 1), where there are relatively short sensilla with thickened bases (statolith-like sensilla). (See arrow no. 2). Bar= l0µm.

£j_g__,____1.Lower reglon of the frontal suture in workers of P. germanica. Promlnent are the sensilla bearing accretlons arranged as 'beads' along the shaft. These accretlons contaln Si. Bar= l0µm.

!J..g___,_1.View of the aggregated accretion in the form of a mushroom in V. oriental ls, seen to protrude from the uppermarglns of the plt the frons plate (Flg. 13), contalnlng Sl in after removal of the encloslng membrane. The the center and Ca at the 'envelope' of an 'mushroom' hangs ln the form of a cluster and organic matrlx The YGs from specimens of the is probably a consolldatlon of secretion same orlgin, l.e. species, caste, age or drug exudlng from this region. The materlal treatment vary but little ln appearance contains Si. Bar= l µm. (Shlmony and !shay, 1981a). However, the YGs from specimens of dlfferent ages are qulte Inside the acoustic box at the end of dlfferent in appearance. Flg. 14 shows the some strings there are aggregatlons contalning pigment granules from a queen pupa of V. Sl (Fig. lla,b). Between the cutlcular layer orlentalis. These granules are elongated­ and the YG layer there are gemmulae-like ovold, not entirely unlform in shape, with a structures contalnlng Caln an organlc matrlx diameter smaller (0.1 µm- 0.2 µm) than in (Fig. 12). adult specimens. They do not fill the entire These structures are more promlnent ln area but, rather, have spaces in between which young hornets than ln adults. Slmllar are apparently filled with a cytoplasmatic structures are present on the outer side of matrix. Inspection of the same area in a

1622 Biomineralization in Social Wasps

fig. 5 a-c. SEMviews of aggregates on the fig. 5 d-f. SEMviews of aggregates on the frons of young hornets. a. K accretion in frons of young hornets. d. Si in organic lower sector of the groove. Bar= 30 µm. matrix outside the groove. Bar= 10 µm. e. b. Same area Si aggregate associated with Ti Sensillum with Si accretion at its base. and Fe. Bar= 30 µm. c. Closeup of Ca Bar= 10 µm. aggregates in the lower sector of the groove. f. Detailed view of Si-Ca complex in organic Bar= 10 µm. matrix. Bar= 10 µm.

freshly-ecloded queen revealed that the diameter. What strikes the eye, however, is pigment granules had undergone thickening to that some of the granules are elongated whereas gradually assume a cylindrical shape. In a others are short. Since the Vespinae are mature queen (several weeks of age) the annual insects, the queen survives for not pigment granules occupying all the available more than one year, wh\le a worker will live spaces are typically cylindrical in shape, at best a number of months. In both aging most commonly 0.6 µm-0.8 µm long and 0.3 µmin queens and workers the yellow color tends to

1623 !shay, J.S., (Benshalom) Sh1mony, T., Arcan, L.

1624 Biomineralization in Social Wasps

Fig. 6a. Presented is a view of the head of a fig. 6d. Offers a magnification of a section hornet displaying the frons region, which is of the inner lining with the delicate the prominent plate in the center of the structure appearing to resemble lacework. photograph. Displayed in the basal part on (Bar= 1D µm) both sides are the bases of the antennae and, in the upper part, the three ocelli. Visible on the frons are large spines 200-6D0 µmin Fig. 6e. A number of strings can be seen. length, and the groove - sutura coronalis in Note that the right one, which had been the center. The entire plate is elevated, detached from its hold on the roof, is now protruding in a wing-like structure towards coiled and abbreviated, with the coiling the middle part of the compound eye. occurring in the narrower parts along its (Bar = 1000 µm) length. Next to this string are two stretched strings with spindle-shaped folds along their length. (Bar= 1D µm)

Fig. 6f. Shows the yellow granules occurring Fig. 6b. Represents a cross-section through between the cuticular layer of the frons the integument, and through the space under­ plate, and the membrane lining the acoustic neath the frons in the region adjacent to the box. These granules are 0.5-0.7 µm long and ocelli. Here one can clearly discern the 0.4-0.5 µmin diameter. K is the dominant groove - sutura coronalis - in the upper part metallic element in the lining membrane, the of the frons, as well as the various setae string and the yellow granules. (Bar= l µm) protrudlngfrom the frons. Visible under- neath are the short strings transversing the 'space' whose length in this region is 100-150 fade (Spradbery, 1973). Fig. 15 shows the µm, with a diameter of 8-15 µm. Most of these pigment granules in a worker over 2 months of strings are straight, although some are age, which had been kept under regular daylight branched, but all lie 50-100 µm apart, bridging illumination. It can be seen that the pigment the inner surface of the frons with the mem­ granules had undergone partial degenerative brane above the protocerebrum. (Bar= lOOµm) changes, evident both lengthwise - usually it is the short ones which occur - and crosswise, where the granules are seen to have lost their typically smooth and rounded appearance, split­ Fig. 6c. Shows a cross-section of the 'space' ting up into smaller subunits. The degener­ along an imaginary band passing through the ative changes revealed a whorl-like structure upper portion of the base of the two antennae. in the inner part of the granules. A similar The space is always ill, i.e., no fluid is to picture can occur in a hornet only 8-10 days be found in this cavity, even in freshly pre­ old, provided that it had been exposed to pared specimens. Visible in the upper part constant illumination throughout its life. center is a cone-like recession (see arrow However, in hornets exposed to daylight for a marked by the letters DP), which is the invagi­ couple of months (i.e., the old ones), there nation of the 'deep pit' in the center of the are no degenerative changes in the pigment frons. From this 'cone' and other parts of the granules if the diet contained one of the roof comprising the inner surface of the frons xanthines (theophylline, caffeine or like plate, strings project which originated from substance). In hornets fed on allopurinol, the inner lining membrane (ILM) of the 'space'. there seemed to be few granules, and those This ILM is a more internal membrane than the that were present showed signs of disinte­ membrane which enfolds a layer of yellow gran­ gration, even though their diameter was at ules. The strings (at least those that did not least twice that of normal hornets. From the break off in the process of fixation) stretch manner of disintegration of these granules, it across the 'space' to its other bank. The would seem that they are helical in structure. maximal height of the 'space' at this region Hornets fed on colchicine are short-lived (not is 600-700 µm while the length of the strings more than 7-9 days), and their pigment granules is proportionate to this height. Clearly are considerably broader (0.5-0.6 µmin dia­ exposed to view are the strings displaying meter), displaying raspberry-like grooves and intermittent thickenings along their length so breaking up differently than In older workers that their diameter ranges between 3-7 µm. (Fig. 16), that is, the breaking up of the The 'roof' of the 'space' is comprised of arcs granules occurs lengthwise rather than cross­ and concavities. The inner lining enfolds the wise, like a log of wood splintering into entire 'space' on all sides while at the bottom longitudinal chips. Hornets that received it comprises a septum separating between the colchicine in their diet together with one of 'space' - henceforth to be designated 'acoustic the xanthines survived similar to hornets box' - and the area below it, i.e., the lnter­ receiving xanthines only (up to several weeks), antennary triangle plate of the hornets. This and their pigment granules did not show the membrane is designated as a separating membrane transformation induced by colchicine alone (SM), traces of which are evident in the photo­ (Ishay et al., 1981). graph. The 'acoustic box' is revealed only In one colony of hornets (out of hun­ after removal of the SM. (Bar= 10D µm) dreds), a pigment aberration was observed in

1625 !shay, J.S., (Benshalom) Shimony, T., Arcan, L. I..!.9..:.....!.TEM cross-section of one of the r\. strings at an area showing an epithelial cell L,' nucleus. (Bar= 2 µm).

~- Transmission electron micrograph (TEM) showing the base of a sensillum and a layer of yellow granules in the frons of a~ oriental is worker. In the center - the base of the sens­ illum. Toward the 11 o'clock position from the center, a section through a nerve fiber is visible (NF). At the periphery of the sen­ sillum base stacked membrane systems (lamellae) are discernible (L). Outside the base the field is loaded with vesicles containing the yellow plgment granules. The borders of three cells and their nuclei (N) are visible; on the Fig. 10. TEMof axon bundle orlginating from top left there is a space left after the min­ the protocerebrum and entering the 'floor' of eral aggregate (dissolved during specimen the 'acoustlc box'. The lndividual axons preparation). (Bar= 4 µm.) penetrate the various strings. (Bar= 2 µm). 8

~- TEMcross-section of one of the strings in the 'acoustic box'. At the perlphery underneath the cuticle layer there ls a tracheal epithelial sheath. A nucleus of this epithelium is shown in Fig. 9. The section reveals two spaces which are separated by a septum. (Bar= 1 µm). Fig. 11 a,.Q. The underlylng surface of the r\. same frons area as in Fig. 5 showing some L,' aggregates. a. Ultrastructure of Si-Ca aggregatlon. Individual granules of Si are easily identifled by EDXwith the organic matrix. Bar= 3D µm. b. Closeup of Si aggregates near the groove area. Bar= 30 µm.

7626 Biomineralization in Social Wasps

Fig. 12. Gemmule-like accretions on the inner side of the frons containing primarily Ca. Bar = l O µm. Fig. 14. Elongated-elliptic pigment granules in a queen pupa of 'L_ orientalis, with large spaces in between. Relative to that of the adult hornets, note the small diameter of the granules, which are still attached to one another. Bar = lµm.

were very short-lived and could only survive under laboratory conditions. The YGs of aberrant workers appeared normal, but where the yellow and brown stripes met, the aber­ rants did not show a sharp demarcation between the granules (yellow region) and the brown cuticle (with melanin) as did normal hornets. Instead, there was a mixture of disorganized fibers which terminated in, or intermeshed with the yellow granules. Photoelectric Properties of the Cuticle As far as the photoelectric properties were concerned the following was noted: after Fig. 13. Side view of the aggregated accretion 10-20 min of illumination, the resistance in in the well of a young worker ('L_ orientalis) the yellow stripes of the cuticle first in­ less than 24 hold. The center of the accre­ creased to a maximal value, then decreased to tion (left) will develop into a form of a a saturation value. This sequence of events 'mushroom'; the main element is Si. The was typical only for the yellow stripes of 'mushroom' is surrounded by 'rods' comprised hornets and wasps in Vespinae but was also mainly of Ca. Bar= 3 µm. encountered in Polistinae. When, after attaining the saturation level, the yellow stripes were subjected to a series of brief the males, namely, their yellow cuticular (30 sec.), alternate exposures to light and stripes lacked the usual lustrous color. darkness, the variations in resistance were Inspection with SEMshows an almost complete clearly periodic - the resistance de- absence of pigment granules in the pertinent creasing when the light was switched on and cuticular region. Instead, there was only a increasing when the light was switched off. faded yellowish color, which did not seem to Irradiation with the light of a He-Ne laser be linked with the granules themselves. The also produced these reversible changes in the ratio of aberrant to normal males on the same resistance. In this case, prolonged initial comb was about l:l. There was no evidence of irradiation was unnecessary and the light of any vital defect nor any impairment of motor the laser was switched off every 10 sec. activity, or of orientation. In another rather than every 30 sec. since the longer colony of workers, a very rare aberration was exposure resulted in irreversible changes. found, consisting of an excess of yellow If a yellow stripe was detached from the pigment in such areas of the body usually body of the hornet (or wasp) and its photo­ colored by the brown cuticle (Fig. 17). The conductive properties measured separately, the ratio of aberrant to normal workers could not properties were invariably seen to be retained be ascertained because the aberrant workers as in the intact hornet, except that the

7627 !shay, J.S., (Benshalom) Shlmony, T., Arcan, L.

Fig. 15. Pigment granules In an old worker over Flg. 17. Macroscopic view from the dorsal two mor.ths of age whose yellow colour had al­ aspect: rlght - control hornet, left - 2 ready faded. Degenerative changes - aberrant workers in whlch the aberration gives dlscernlble ln the granules - entail external rlse to the appearance of a yellow color In lrregularltles, a partial breakup and the usual regions, although ln wlder strips abbrevlatlon of length, although the diameter than normal, as well as ln segments usually ls larger than usual. Bar= lµm. only covered by brown cuticula (ln the abdominal and cephalic regions as lndlcated by arrows). In addltlon to the color changes there are also malformations in the alea and pedes.

transparent cutlcula per se ls not photo­ conductive. In addition, very high values of resistance were obtained, on the order of 109-1010ohm, as compared to measurements on whole yellow stripes where the obtained values were on the order of 107-108 ohm, I.e., a 10-100 fold difference ln resistance. The resistance values obtained on measuring the photoconductlvity of the YGs removed from the cuticle were about the same as In entire yellow stripes (i.e., including the cuticle), attaining up to 107-108 ohm. However, llke in measurements of the transparant cutl­ cule alone, the resistance stabilizes at a fixed level and the YGs here show better photo­ conductlve properties than thelr counterpart Flg. 16. L orlentalls worker which had Intact yellow stripe. Evaluation of the re­ received colchlclne ln the dlet. The following sistance as a function of wavelength for UV, changes ln the pigment granules are discernible violet, red and IR light as compared to white compared to the control group: Here they are llght was also Investigated. We found that shorter, wlth a considerably larger diameter the UV light produced practically the same (0.53-D.66 µm), ln an advanced stage of break­ changes In resistance as the white llght. The up dlsplaylng raspberry-like grooves. spectral dependence of the photoconductivity Bar= l µm. ln the yellow stripes following prolonged lllumlnation (15-20 mln) was similarly Investi­ gated. It was found that the maximumof the lnltlal resistance attained during the satur­ spectral_distrlbution corresponded to a wave-­ ation period was higher. Measurements taken length of approximately 420 nm. This result on the transparent cutlcula alone, that is, gave the activation energy(~ 2.9 eV) necessary following removal of the yellow granules to obtain the transition of carriers from the together wlth the base membrane, showed that valence band to the conduction band in semi­ the resistance ls constantly on the rlse, both conductors (see Hannay, 1959; Ryvkin, 1964; ln llght and ln darkness, meaning that the Kittel, 1968; Watson, 1969).

1628 Biomineralization in Social Wasps

A mathematical model (!shay et al., 1980) fits the Arrhenius law with activation energies for the relative changes in resistance in the (Ea) = l.0-2.2 eV. The effect of diet on the yellow stripes as a photoconductive process electrical resistivity to the direct current conforms to the general model for a semicon­ of the yellow and brown stripes of the cuticle ductor with traps. For these materials the of hornet workers was measured in the dark electrical conductivity seems to be due to within a temperature range of l0-32°C. It was both negative and positive carriers. As a found that diet exerts a significant effect result of releasing an electron, a chemical both on the level of the electric resistivity bond is opened, i.e., a hole is formed of the cuticle as well as on the shape of the (yielding traps). Such holes can be filled by resistivity 'line'. Neither age nor pigment neighboring electrons which in turn will open exerted any significant influence. On ordi­ new holes, etc. This movement of electrons nary diet alone, a hysteresis loop was obtained filling neighboring holes contributes to the between the heating and the cooling lines, electrical conductivity in the granule. In which pointed to the effect of memory (in the our case, when illuminating the yellow stripes cuticle). Feeding on allopurinol or theophyl­ of the hornet, a saturation level had to be line resulted in a straight 'line' of resist­ reached first where all the traps were filled. ance, i.e., there was an improvement of the Only then could additional electrons contribute semiconductive properties of the cuticle, al­ to conductivity. In darkness the reverse though this somehow interfered with the memory holds true, i.e. carriers move from the effect produced by feeding them on a regular conductance band to the valence band, hence, sugar solution diet (Rosenzweig et al., 1985). conductivity decreases. Our final model was The thermoelectric (Seebeck) coefficient well supported when we observed the measured (S = 6V/6T) in various cuticular areas of the data on 8 different hornets. Oriental hornet (Vespa orientalis) fluctuates The photoconductive properties are affected from 0•3 to 2•4 mVdeg-I within a temper- by drugs. Thus, feeding on xanthines improved ature range of 27-36°C and when the temperature these properties, as evidenced from the fact difference between the two measuring electrodes that a) the baseline of resistance was re­ (6T) is 0•6-to 8•0°C. The values measured tained throughout the measurement; b) the on the brown-colored cuticle suggest an ~-type amplitude obtained between the resistance in conduction, while those measured on the yellow­ light and that in the dark did not change colored cuticle point to a R-type conduction. significantly after reaching the base level. It is suggested that hornets use this pheno­ There was also a marked difference between menon for temperature detection. hornets kept in the dark on xanthine (caffeine) The electric capacitance in the integument in their regimen, and those living in darkness of the Vespa orientalis was investigated and without caffeine. In the former, the photo­ found to be dependent on various factors. These conductive properties were marked and quite included: biological factors such as age, similar to those in control hornets kept under region of integument and the predominant illumination. In allopurinol-fed hornets there pigment in the measured region, as well as was an abbreviation of the saturation time (to physical factors. At 1000 Hz the mean capaci­ about 10 min), whereas feeding on colchicine tance was about 0.6 nF as opposed to 3.0 nF at provoked a deterioration of the photoconductive 100 Hz. This inverse relationship between fre­ properties (!shay and Shimony, 1982). quency and the electric capacitance might Thermoconductivity of the Cuticle point to the presence of polar substances. The The yellow and brown stripes on the capacitance decreases under illumination and Vespinae cuticle are thermoconductive. The increases in the dark. With an increase in response of the cuticle to temperature changes temperature there is at times an increase and is fairly stereotypic, entailing the following at other times a decrease in the capacitance, stages: a) a sharp drop in the resistivity, which is inversely correlated to the resistivity. with a rise of temperature up to values at which hornets naturally become active (around Discussion 24°C, vid. Bodenheimer, 1933). A reverse pattern of an increase in the electrical To the best of our knowledge, the struct­ resistance of the cuticle with a decrease in ural conglomerate described herein is most temperature is also found at temperatures down likely an organ which is new to science. to -35°C. At around 24°C the electric resisti­ It is probably a combination of an acoustic vity of the hornet cuticle reaches nadir. and equilibratory organ, similar in some re­ b) Within the range of about 27-32°C, a spects to an organ in various vertebrate plateau-like phase is obtained in the course . The 'space' which we have described of which no further significant change in the in the present paper is apparently an 'acoustic resistivity with a rise in temperature took box', whereas the strings serve to inwardly place. These results point to the fact that transmit sound waves or vibrations (the so­ at the lower temperatures the cuticule behaves called airborne and solid-borne sounds) as like a semiconductive material while at the well as data on spatial orientation, all of higher temperatures the cuticle displays quasi­ which are 'forwarded' in the direction of the metallic conductance with phase transition brain, probably to the areas in the protocere­ (Gutmann and Lyons, 1967). The temperature brum. The 'acoustic box', sealed from all dependence in darkness and low temperatures sides, contains strings which are long and

1629 I shay, J.S., (Benshalom) Shimony, T., Arcan, L. thin in the lower part of the 'box' but which Fig. 18_:A Schematic Model of the Tympanic Organ become shorter and thicker towards its upper part near the ocelli. The tegumentary outer I Assumed Function ) I The functioning Organ I plates and their attached structures - the setae, the delicate cuticle at their base, and Mechanical Pressure Pit Well Setae and Epi­ the layer of YGs and the membrane which seals Sensed Membrane Crystals uticle the deep pit - all act as components of a at their tympanic membrane. Bases The Model. ' j Tentatively, we speculate that auditory sensation and/or mechanical pressure may be picked up externally and transmitted intern­ jTympani c Membrane'I The membrane at the ally in the following manner: the setae on Base of the Setae the frons pick up sound waves or contact pressure or air motion and transmit the re­ l l sultant impact via the cuticle layer at their base, which acts as a tympanic membrane, to Piezoelectric The Yellow Pigment the layer of YGs containing the metallic Transducers Granular La ers element K where the mechanical pressure is translated into electric energy due to the (Mechanical piezoelectric properties of the granules (J.S. to !shay, unpublished observations). In this case Electrical The Lining Layer Inside the YGs probably act as piezoelectrlc trans­ and The Acoustic Box with ducers which convert sound waves into alter­ Viceversa) its Plexuses and Crystal nating electric current (see Mason, 1966; Cope, A re ates 1975). As far as electrical conducting ele­ ments, their piezoelectric properties and other ! 1 structures both inside and outside the frons plate are concerned, the 'deep pit' contains Amplification of The Strings granules of Si, K, Ca, Ti and Fe (the presence Signals and Inside the of Ti and Fe in pigments has already Transmission Acoustic Box been previously described, e.g., Kikkawa et al., 1955). These granules press upon the 1 ! cuticle, which projects inwardly into the 'acoustic box', and are probably associated Signal Receptor with the spatial orientation of the hornet. The Filtering and Neuropil in the lining layer inside the acoustic box with its Transduclng and Protocerebrum plexuses containing Ca in an organic matrix Demodulatinq converts electric energy into mechanical energy where the latter is amplified and, following filtration, is transmitted as vibrations via The membrane-ensheathed, cyst-like deep pit in the strings (in some of which are aggregations the frontal area, with its secretion aggregates containing Si) to the sensitive areas in the of Si, K, Ca Ti, Fe, largely resembles a stato­ protocerebrum. In our opinion, such conversion cyst. On general lines, this parallels, the of energy types is possible because hornet and situation in decapod Crustaceans (Lang and wasp cuticle possesses organic semi-conductor Yonge, 1935; Schone, 1971) because the deep pit properties, found not only in the head region with the metallic aggregation within it is but also in other areas such as the abdomen situated between the two antennae, which are (see schematic presentation of the model). homologous to the antennules in crustaceans The cuticle is also endowed with photoelectric (Meglitsch, 1972). We contend that the pit properties (Croitoru et al., 1978; I shay and can be regarded, at least in its lower portion, Croitoru, 1978; I shay et al., 1980), thermocon­ as analogous to the area at the base of the ductivity (!shay et al., 1982 b, c; !shay and antennules in crustaceans which has fused, in Shimony, 1983), thermoelectriclty (Seebeck the course of evolution, to form a single effect - see Shimony and !shay, 1981b), piezo­ statocyst at the center of the frons - an electricity (J.S. !shay, unpublished observa­ ideal site for the organ involved in stabil­ tions) and, finally, an electrical capacitance ization and equilibrium (!shay et al., 1983). (Shimony and !shay, 1984). Other biological But, unlike the situation in crustacean Oeca­ materials are similarly known to possess piezo­ poda where the mineral grain is introduced by electric properties, to wit: a) the bones the from the surroundings available, the (Bassett and Becker, 1962; Shamos and Lavine, Vespinae secrete these minerals both on the 1967; Cope, 1975), and b) otoliths (Morris and inner and the outer side of their cuticles. Kittleman, 1967) in bony fishes where the Since in the nest of Vespinae various otoliths contain equal amounts of Ca and Na sounds are produced both for purposes of (although no K was found), and c) the skeleton communication as well as in the course of of various (Zilberstein, 1972). routine nest activity, it seems reasonable (See Schematic Model of the Tympanic Organ, that an organ with an acoustic box such as is Fig. 18). described above should develop, especially

1630 since hornets and wasps engage in comb building become shorter and lose their characteristic mainly in darkness and since, both under natu­ shape with increasing age of the insect, by ral as well as experimental (special centri­ their constant exposure to illumination, or to fuge) conditions, the building is directed certain drugs. Under the effect of colchicine, towards the gravitational force (!shay, 1975, for instance, there are degenerative changes 1976 a, b; !shay and Sadeh,1975, 1977). The of the pigment granules' diameter at a faster building process itself, entailing the for­ rate than that due to aging and its subsequent mation of hexagonal cells, greatly relies not breakup into subunits. Interestingly, the only on the gravitational sense, but also on feeding of caffeine (or purine) together with the mechanoreceptor one. Thus hornet workers colchicine prevents the breakdown of the tap with their antennae on each fresh layer of granules without altering the longevity of the the wall being built, probably to ascertain insect. Apparently the various xanthines in static conditions such as strength, thickness, some way prevent the destruction of the and the degree of uniformity (!shay et al., membranoussystem of the granules by 1967, 1982a). We believe that the structure colchicine. The mode of action of xanthines discovered by us conforms functionally to a in hornets is as yet unclear. In addition to mechanoreceptory organ in which the different the phenomena associated with elevated cAMP length, diameter and spindle-shape of the levels, it is possible that the stereo­ strings in their various areas enable electronic structure of the metabolized selective transmission of sensory messages. methylated alkaloid partially resembles that It is possible that the 'acoustic box' with of juvenile hormone (as in the distance its strings had developed from a dilatation of between the two methyl groups and the keto a large trachea. group on the pyrimidine ring). It might Mechanoreceptors have been described in therefore act to block the receptor to juvenile various insects (Haskell, 1961; Busnell, 1963; hormone. The disappearance of the YGs caased Dethier, 1963; Schwartzkopff, 1974), where by feeding with allopurinol is to be attributed they are associated with the base of hairs on to its inhibitory activity on the enzyme xan­ the joints or occur as an organ within an thine oxidase; whereby the oxidation of various enlargement of a trachea in a leg, in the pterins is inhibited and consequently there is abdomen or in the base of the antennae no formation of such products as xanthopterin, (Johnston's organ). In larvae and adults of isoxanthopterin, and leucopterin, (all of which Ephemeroptera there are certain organs which are dyes). In other words, there is inhibition are apparently gravity receptors in the center of the formation of pigment granules from unco­ of the head (Gross,1903; Wodsedalek, 1912). lored precursors. These and like pigments were These have been named Palmen's organ and are detected in hornets (Ikan and !shay, 1967). cuticular secreted nodules at the juncture of Mutant hornet males lacking the yellow pigment four trachae on the mid-dorsal line just also lack the oxidized derivatives (which are behind the eyes. present in the normal males), which suggests that As for the yellow pigment granules: their the mutation relates apparently to a deficiency general appearance resembles the pterinosomes, or impairment in the activity of xanthine oxidase as described from the amphibian~ cinerea (Croitoru et al., 1977). Conversely, the pre­ (Bagnara and Hadley, 1973), and from fishes sence of excess yellow pigment stems probably (Matsumoto and Obika, 1968). These pterino­ from overactivity of this enzyme at the expense somes are spheroid in shape and composed of of melanin. Bagnara et al. (1979) in vertebrates concentrically arranged membranes, with the and Schliwa and Euteneur (1979) have reported pigment (pteridines) enclosed within the inner that in invertebrates pigment granules contain membranes. In invertebrates, tegumental pig­ several types of pigment like purines,pteridines, ment granules have been observed in the wings melanins, and others. of butterflies, in Odonata and in Cnustacea The yellow pigment granules (organelles) in (Descimon, 1965; Green and Neef, 1969; Veron social wasps deserve to be called xanthosomes et al., 1974). With age there are changes both because they are located within chromatophores in the density and the size of the granules; that contain~ yellow pigment, apparently of therefore, it appears that development of the one single type (xanthophores). These pigment granules proceeds in several stages, one of granules are apparently fixed more or less in which entails the formation of a new granule their position, without much degree of freedom, and its enlargement by growth within the inter­ in contrast to those reported in Odonata (Veron spaces between the membranes. Participating in et al., 1974), where some pigment bodies migrate the formation of such granules are the Golgi during intermediate phases of color change. apparatus and the endoplasmic reticulum (Toda The brown stripes of the cuticle, respond and Fitzpatrick, 1972; Bagnara, 1972). Our similarly to the yellow stripes, to various phy­ findings suggest that the granules develop in sical treatments like photo and thermoconduc­ synchrony with ontogeny of the insect from tivity, i.e., they are endowed with the same or the pupal stage to somewhat beyond the similar electrical properties, but with one noti­ eclosion of the imago. Their final dimensions ceable difference: these properties are not are dependent on, or determined by, the insect influenced (or are less influenced) by the caste (worker or queen), or its sex (female or different drugs used by us in the various male) where there are, of course, also species­ specific differences. The granules gradually

1631 Ishay, J.S., (Benshalom) Shimony, T., Arcan, L. experiments. As we have shown, the drugs tended 5. Becker E. (1937). Uber das Pteridinpigment to influence the integrity, size or shape of the bel lnsekten und die Farbung und Zeichnung yellow pigments, and the resultant changes were von Vespa ln besonderen. Z. Morph. Okol. usually correlated with photo- or thermocon­ Tiere 32, 672-751. ductivlty. 6. Bodenheimer FS. (1933). Uber die Akivitat The fact that there are two types of cuti­ von Vespa orientalls in Jahresverlauf in cular lnteractlon with light - in one of which Palastina. Zool. Anz. ill, 135-140. the resistance rlses up to a saturatlon value, i.e.,positive photoconductivlty, while in the 7. Busnell RG. (ed.) (1963) Acoustic other the cuticle lmmediately behaves as a Behavlor of Anlmals. Elsevler, New York, photoconductor, i.e., negative photoconductl­ 933 pp. vlty - leads to the conclusion that lt ls 8. Chapman RF. (1969). The Insects Structure essential to verify which energy of the lncl­ and Function. English Universitles Press dent light lnduces the rise in the resistance Ltd., London, 819 pp. and which energy gives the transition of car­ riers when the yellow stripe behaves as a photo­ 9. Cope FW. (1975). A review of the conductor. By correlating the increase in re­ appllcation of solid state physics sistance with the wavelength of the lncldent concepts in biological systems. J. Blol. light it was found that UV light is responsible Phys. ;!, 1-41. for that increase, whereas the photoconductivity 10. Croitoru N, Ishay JS, Perna B, Ikan R. which occurs after the saturation is dependent (1977). Photoelectric properties on the on violet and blue light. These findings yellow stripes of the social wasp cuticle. suggest that molecular changes must take place The XI Scientific Meeting of the because of the energies involved. A lower Association for the Advancement of Sclence energy of the light is responslble for free in Israel: p. 69; Dept. Chem. Bar-Ilan carrlers In the photoconductlon process. Unlv., Ramat Gan. We may therefore assume that the electrl­ cal conductivity, as well as other properties 11. Croitoru N, Ishay JS, Arcan L, Perna B. of the yellow stripes, are due to the contained (1978). Electrical resistance of the yellow YG. We believe that the described tympanic stripes of soclal wasps under lllumination. organ in the hornet head is 'involved' in geo­ Photochem. Photobiol. 28, 265-270. troplc orientation, whereas such yellow pigment 12. Desc1mon H. (1965). Ultrastructure et as is dlspersed throughout the body lntegument pigmentation des ecailles des is 'lnvolved' In phototaxis, and enables the Lepidopteres. J. Microscopie !, 130. conversion from phototaxis to geotaxis in these animals, a phenomenon assumed to occur in all 13. Oeth1er VG. (1963). The Physiology of Pterigota (Llnsenmair-Ziegler, 1970). Insects Senses. Methuen and Co. Ltd., London, 266 pp. Acknowledgments 14. Edwards R. (1980). Social Wasps. Their biology and control. Rentokll Llmlted, East We thank Mr. Jacob Delarea for his excel­ Grinstead, 398 pp. lent technlcal assistance in the electron mlcroscopic study, and Ms. Stella Levi-Weitman 15. Green JP, Neef MR. (1969). Fine structure for her time and help in preparing the manuscript. of the fiddler crab epldermis. Blol. Bull. ill, 401. References 16. Gross J. (1903). Uber das Palmensche Organ der Ephemeriden. Zool. Jb (Anal). li, l. Bagnara JT. ( 1972). Interrelationship of 91-106. melanophores, lridophores, and xanthopores, in: Plgmentation: Its genesis and biologlc 17. Guiglia D. (1972). Les Guipes Sociales control, V. Riley (ed), Appleton-Century­ (Hymenoptera: ) d' Europe Craft, NY, 171-180. Occldentale et Septentrionale. Masson et Cle, Parls, 181 pp. 2. Bagnara JT, Hadley ME. (1973). Chromatophores and color change. Prentice­ 18. Gutmann F, Lyons E. (1967) Organic Hall Inc. Englewood Cliffs, NJ, 202 pp. Semiconductors. John Wiley and Sons, New York, 858 pp. 3. Bagnara JT, Matsumoto J, Ferris W, Frost SK,Turner Jr WA,Tchen TT, Tayler JO. 19. Hannay NB. (1959). Semiconductors. (1979). Commonorigin of pigment cells. Reinhold Pub. Corp. New York, 42 pp. Science 204, 410-415. 20. Harmsen R. (1966). A quantitative study of 4. Bassett CAL, Becker RO. (1962). Generation the pteridines in Pieris brassicae L. of electrlc potentials by bone in response during post-embryonic development. J. to mechanlcal stress. Science 137, Insect Physlol. 11., 9-22. 1063-1064. - 21. Haskell PT. (1961). Insect Sounds. H.f. and G. Witherby Ltd., London, 189 pp.

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Editor's Note: All of the reviewers' concerns were appropriate­ ly addressed by text changes, hence there is no Discussion with Reviewers.

1634